RU5 of Mason-Pfizer monkey virus 5' long terminal repeat enhances cytoplasmic expression of human immunodeficiency virus type 1 gag-pol and nonviral reporter RNA

Abstract

Retroviruses utilize an unspliced version of their primary transcription product as an RNA template for synthesis of viral Gag and Pol structural and enzymatic proteins. Cytoplasmic expression of the gag-pol RNA is achieved despite the lack of intron removal and the presence of a long and highly structured 5' untranslated region that inhibits efficient ribosome scanning. In this study, we have identified for the first time that the 5' long terminal repeat (LTR) of Mason-Pfizer monkey virus (MPMV) facilitates Rev/Rev-responsive element-independent expression of HIV-1 gag-pol reporter RNA. The MPMV RU5 region of the LTR is necessary and directs functional interaction with cellular posttranscriptional modulators present in human 293 and monkey COS cells but not in quail QT-6 cells and does not require any viral protein. Deletion of MPMV RU5 decreases the abundance of spliced mRNA but has little effect on cytoplasmic accumulation of unspliced gag-pol RNA despite complete elimination of detectable Gag protein production. MPMV RU5 also exerts a positive effect on the cytoplasmic expression of intronless luc RNA, and ribosomal profile analysis demonstrates that MPMV RU5 directs subcellular localization of the luc transcript to polyribosomes. Our findings have a number of similarities with those of reports on 5' terminal posttranscriptional control elements in spleen necrosis virus and human foamy virus RNA and support the model that divergent retroviruses share 5' terminal RNA elements that interact with host proteins to program retroviral RNA for productive cytoplasmic expression.

FIG. 1.

Summary of the structures of the gag and luc reporter plasmids. Shown in light gray are the U3, R, and U5 regions of the MPMV LTR; shown in white are the U3, R, and U5 regions of the SNV LTR; 5′-terminal oval, CMV immediate-early (IE) promoter-enhancer; dark gray rectangles, HIV gag-pol coding region; 5′ and 3′ ss, splice sites; white rectangles, luc coding regions; and 3′ terminal oval labeled “p(A),” polyadenylation signal.

FIG. 2.

RPA of total cellular RNA from transfected QT-6 quail cells verifies reporter RNA synthesis. (A) Relationship among the gag-pol reporter plasmid, the uniformly labeled antisense runoff HIV-1 5′ UTR RNA probe, and protected unspliced and spliced transcripts with sizes indicated. (B) Forty-eight hours posttransfection total RNA was isolated and treated with DNase. Twenty micrograms was subjected to RPA with uniformly labeled antisense HIV-1 5′ UTR and gapdh RNA probes and polyacrylamide gel electrophoresis. Labels indicate the reporter plasmid, RNA preparation, and protected transcripts.

FIG. 3.

RPA of nuclear and cytoplasmic RNA from transfected 293 cells reveals that deletion of MPMV RU5 does not reduce the steady-state unspliced gag RNA level but does reduce the spliced RNA level. Forty-eight hours posttransfection RNAs were isolated and treated with DNase. Nuclear (15 μg) and cytoplasmic (25 μg) RNAs were subjected to RPA with uniformly labeled antisense 5′ UTR and gapdh RNA probes and polyacrylamide gel electrophoresis. Labels indicate the reporter plasmid, RNA preparation, and protected transcript. (A) Analysis of pSVgagpol-rre reporter RNA in the presence or absence of pRev. (B) Analysis of MPMV LTR and MPMV ΔRU5 reporter RNA.

FIG. 4.

Northern blot of nuclear and cytoplasmic RNA from transfected 293 cells reveals that MPMV RU5 has minimal effect on the steady-state luc RNA level or cytoplasmic accumulation. Forty-eight hours posttransfection RNAs were isolated and treated with DNase. Ten-microgram aliquots were subjected to electrophoresis on a 1.2% agarose gel with 5% formaldehyde, transferred to a Duralon-UV membrane, and hybridized to α-³²P-labeled DNA probes complementary to luc and cp. The blots were subjected to PhosphorImager analysis. Labels indicate the reporter plasmid and RNA fraction.

FIG. 5.

Northern blot of luc reporter RNAs across the ribosomal profile reveals that MPMV RU5 redistributes cytoplasmic RNA to the light and heavy polyribosomes (LP and HP). (A) Representative ribosomal profile of transfected 293 cells. Cytoplasmic extracts were subjected to sucrose gradient ultracentrifugation, fractionation, and spectrophotometry (A₂₅₄). Positions of the 60S ribosomal subunit, 80S monosomes, light polyribosomes (two to three ribosomes), and heavy polyribosomes (four to six ribosomes) are indicated. The arrow indicates the direction of the gradient. (B) Each fraction was subjected to electrophoresis on a 1.2% agarose gel with 5% formaldehyde, transferred to a Duralon-UV membrane, and hybridized to a α-³²P-labeled RNA probe complementary to luc. RNA levels were quantified by PhosphorImager analysis. Labels indicate the reporter plasmid and the fraction of the ribosomal profile. (C) Shown is quantification of luc RNA levels across the ribosomal profile, which were tallied and expressed as percentages of total PhosphorImager units. ⋄, MPMV luc; ▪, MPMV ΔRU5 luc.